PROJECT SUMMARY/ABSTRACT: There is a fundamental need to understand how transcription factors function together with their co-regulators to redefine a cell?s developmental potential through cell fate transitions. Our current understanding is limited to a small number of examples often based on simple linear pathways with a transcription factor upstream of its co-regulators either activating or repressing an enhancer, which in turn regulates its cognate gene in time and space. However, without a better understanding of the mutual dependencies between transcription factors and their co-regulators including epigenetic enzymes and collaborative DNA binding factors, it will be impossible to predict how manipulation of either will influence cell fate and developmental potential. The long-term goal of the lab is to understand all levels of molecular control of cell fate transitions in order to efficiently reprogram cells to desired phenotypes. The objective here is to focus on a transcription factor, Foxd3, which is essential to maintain the developmental potential of various stem cells. The central hypothesis is that Foxd3 is used iteratively in stem cells to redefine the cell?s development potential by establishing poised enhancers in association with its co-regulators and collaborative cell specific transcription factors. This hypothesis derives from preliminary data establishing a dual functional role for Foxd3 as a simultaneous activator and repressor through its interaction with and regulation of multiple epigenetic factors. As such it poises genes and redefines the developmental potential of different stem cell populations by moving to new enhancer sites. The following specific aims are proposed: 1) Determine the epistatic relationship between Foxd3, H3K4 methylation, nucleosome depletion, and H3K27 acetylation, 2) Uncover the mechanistic basis for Foxd3 movements during the embryonic stem to epiblast cell transition, 3) Identify role of Foxd3 in cohesin recruitment and enhancer- promoter looping during developmental gene activation. In aim 1, epistasis and structure-function analyses will be performed using mutants of the Foxd3 and its coregulators to determine the interdependencies between the factors in establishing a dual-functional complex at bound sites. In aim 2, post-translational modifications and collaboration with other transcription factors will be evaluated to dissect the mechanistic basis of Foxd3 movements. In aim 3, Foxd3?s role in cohesin recruitment and enhancer-promoter looping will be evaluated using time-course and epistasis experiments. The proposal is highly significant as it will provide novel paradigms of gene control central to a cell?s developmental potential. These paradigms are unlikely to be specific to Foxd3, but rather reflect general strategies used by stemness transcription factors to retain or induce a stem cell?s full potential. Such knowledge will allow for better-designed strategies for cell manipulation and improved ability to predict effects of therapeutics aimed at the epigenetic co-regulators.